T lymphocytes are involved in most specific immune defenses against microorganisms, parasites and toxic substances. In addition, they play pivotal roles in allergy, autoimmunity and the rejection of surgical transplants. These cells thus contribute in both positive and negative ways to many disease states. The long term goals of this proposal are to understand the molecular mechanisms of antigen recognition by human T lymphocytes, and to chemically define the role that products of the Major Histocompatibility Complex (MHC) play in this process. At present we have some understanding of the components involved but almost no appreciation of the way they work or interact with each other. The response of cloned human cytolytic T cells (CTL) to allogeneic class I antigens (HLA-A,B) will be studied as this represents a simplified system for which key reagents, i.e., cloned genes, purified HLA-A,B antigens and well defined monoclonal antibodies, are available. Two distinct strategies encompassing three specific aims are proposed. In aim 1 in vitro mutagenesis will be used to produce HLA-A,B molecules that have alterations in regions that have been conserved during evolution. Their structural and functional properties as targets for allogeneic CTL will be examined. This will identify the "active site" of the HLA-A,B molecule for productive interaction with any T cell that is "restricted" through these MHC molecules. Aims 2 and 3 will first, define an assay to measure the interaction of target HLA-A,B antigen with the surface of specific CTL and, second, use this assay to develop a completely defined in vitro system in which the interaction can be studied. As the interactions to be studied are of low affinity the methods to be used to detect binding will amplify effective or functional affinities by the construction of defined multivalent ligands using monoclonal antibodies against HLA-A,B and the variable T cell antigen receptor. This proposal aims to provide insight into a fundamental immunological process. It may provide formal proof of the identity of the variable antigen receptor of T cells and define in molecular terms the function of HLA-A,B. The methods developed may result in better assays for T cell responses that may have application in diagnosis in many clinical situations, particularly those involving the transplant recipient.